PROJECT SUMMARY Cutaneous squamous cell carcinoma (CSCC), the most aggressive non-melanoma skin cancer, is the second most common cancer in the USA and worldwide. Head and neck SCC (HNSCC) is a leading cause of cancer deaths worldwide and accounts for 3-5% of all cancers in USA. Together, these cancers (SCC) contribute substantially to morbidity and mortality. Our long-term goal is to develop novel approaches to SCC prevention and treatment based on a deep understanding of its pathogenesis. The specific goal of this application is to define a novel tumor suppressive role for alkaline ceramidase 1 (ACER1), a member in the alkaline ceramidase family that we identified initially from the yeast Saccharomyces cerevisiae and then from mammals, in SCC development and progression. SCC arises from keratinocytes or squamous cells through a multistage process including tumor promotion. Tumor promotion alters the expression of genes controlling cell proliferation, differentiation, and apoptosis and identification of such genes offers targets for chemoprevention and/or therapy. Through gene expression profiling, we find that 12-O-tetradecanoylphorbol-13-acetate (TPA), the classic tumor promoter, inhibits the expression of ACER1 in normal epidermal keratinocytes. Importantly, we demonstrate that overexpression of the human ACER1 transgene in the mouse epidermis increases tumor latency and reduces tumor burden in the two-stage skin carcinogenesis model initiated with 7,12- dimethylbenz(a)anthracene (DMBA) and promoted with TPA, suggesting that ACER1 downregulation contributes to tumor promotion. Consistent with this notion, we show that ACER1 expression is markedly suppressed in SCC cell lines and tissues and that restoring ACER1 expression inhibits the proliferation of SCC cells, suggesting that ACER1 downregulation leads to the hyperproliferation of premalignant and malignant keratinocytes, thereby promoting SCC development. Based on these important findings, we hypothesize that ACER1 acts as a tumor suppressor in SCC and that its downregulation promotes skin tumorigenesis by promoting epidermal keratinocyte hyperproliferation. As a further corollary, we hypothesize that rectifying the ACER1 mediated pathway will inhibit skin tumorigenesis. These hypotheses will be tested by two specific aims. In Aim 1, we will define the tumor suppressive role for ACER1 in SCC using both in vitro model of tumor promotion and animal models of skin tumorigenesis and establish that dysregulation of the ACER1/sphingolipid pathway is a predictive biomarker for early onset SCC. In Aim 2 we will define the cellular and molecular mechanisms by which the ACER1/sphingolipid pathway functions as a tumor suppressor in SCC. The proposed research will provide not only insights into the role of the ACER1/sphingolipid pathway in SCC suppression but also an intellectual framework for skin cancer prevention and treatment.